1. Field of the Invention
The invention belongs to the field of protein production in prokaryotic cells.
The invention relates to methods for the production of recombinant DNA-derived heterologous protein in prokaryotic cells, wherein said heterologous protein is secreted extracellularly as an active and correctly folded protein, and the prokaryotic cell contains and expresses a vector comprising the DNA coding for said heterologous protein operably linked to the DNA coding for the signal peptide OmpA.
2. Related Art
Prokaryotic expression systems for heterologous proteins are commonly used for proteins which do not require mammalian glycosylation patterns as they provide a cheap way of producing large quantities of said protein. The formation of highly aggregated protein or inclusion bodies can be commonly found in high-level expression of many heterologous proteins in E. coli. One way of protein production is via inclusion bodies which develop in cytoplasm. Cell wall and outer membrane components of the prokaryotic cells used for production (e.g. E. coli) usually contaminate the cell lysate containing the heterologous protein when said inclusion bodies are prepared by low-speed centrifugation. The outer membrane component can be eliminated by selective extraction with detergents and low concentrations of either urea or guanidine.HCl.
One example of such a heterologous protein is a tPA derivative. Tissue plasminogen activator (tPA) is a polypeptide containing 527 amino acid residues (Pennica, D., et al., Nature 301:214-221 (1983)) with a molecular mass of 72 kDa. The molecule is divided into five structural domains. Nearby the N-terminal region is a looped finger domain, which is followed by a growth factor domain. Two similar domains, kringle 1 and kringle 2, are following. Both finger and kringle 2 domains bind specifically to the fibrin clots thereby accelerating tPA protein activation of bound plasminogen. Downstream of kringle 2 is the serine protease, with its catalytic site located at the C-terminus. The serine protease is responsible for converting plasminogen to plasmin a reaction important in the homeostasis of fibrin formation and clot dissolution. The correct folding of tPA requires the correct pairing of 17 disulfide bridges in the molecule (Allen, S., et al., J. Biol. Chem. 270:4797-4804 (1995)).
Clinically, tPA is a thrombolytic agent of choice for the treatment of acute myocardial infarction. It has the advantage of causing no side effects on systemic haemorrhaging and fibrinogen depletion (Camiolo, S. M., et al., Proc. Soc. Exp. Biol. Med. 38:277-280 (1971)). Bowes melanoma cells were first used as a source in tPA production for therapeutic purposes (Griffiths, J. B., and Electricwala, A., Adv. Biochem. Eng. Biotechnol. 34:147-166 (1987)). Since a consistent process with efficient production of highly purified protein in good yield is required for clinical use, the construction of full-length recombinant-tPA (r-tPA) progressed to mammalian cells. Chinese hamster ovary cells were transfected with the tPA gene to synthesize the r-tPA (Cartwright, T., “Production of t-PA from animal cell culture,” in Spier, R. E. and Griffiths, J. B., eds., Animal Cell Biotechnology, Vol. 5., Academic Press, New York (1992), pp. 217-245; Lubiniecki, A., et al., “Selected strategies for manufacture and control of recombinant tissue plasminogen activator prepared from cell culture,” in Spier, R. E., et al., eds., Advances in animal cell biology and technology for bioprocesses, Butterworths, London (pp. 442-451). The recombinant product produced by a mammalian fermentation system was harvested from the culture medium. Attracted by simplicity and economy of production, a number of efforts in producing r-tPA from bacteria, especially from Escherichia coli, were investigated (Datar, R. V., et al., Biotechnology 11:349-357 (1993); Harris, T. J., et al., Mol. Biol. Med. 3:279-292 (1986); Sarmientos, P., et al., Biotechnology 7:495-501 (1989)). Regarding the low yield and the formation of inclusion bodies, which resulted in misfolding and in an inactive enzyme, numerous strategies have been proposed to overcome these problems. The major criterion is to synthesize the smallest molecule, which is still active instead of full-length tPA.
Several deletion-mutant variants including kringle 2 plus serine protease (K2S) were considered. However, the enzymatic activity of the recombinant-K2S (r-K2S) was obtained only when refolding processes of purified inclusion bodies from cytoplasmic compartment were achieved (Hu, C. K., et al., Biochemistry 33:11760-11766 (1994); Saito, Y., et al., Biotechnol. Prog. 10:472-479 (1994)). In order to avoid the cumbersome refolding processes and periplasmic protein delivery, special bacterial expression systems were exploited (Betton, J. M., et al., J. Biol. Chem. 273:8897-8902 (1998); Scherrer, S., et al., Appl. Microbiol. Biotechnol. 42:85-89 (1994)). Despite periplasmic expression of tPA, overexpression led to inactive aggregates, even in the relatively high oxidizing condition in the periplasm.
In the prior art, there are a few descriptions of methods for the preparation of recombinant K2S in E. coli. However, there is no disclosure of a method leading to a cost effective method for large scale production of biologically active K2S.
Obukowicz, M. G., et al., Biochemistry 29:9737-9745 (1990), expressed and purified r-K2S from periplasmic space. The obvious disadvantage of this method was an extra periplasmic extraction step, which is not suitable for large scale production.
Saito, Y., et al., Biotechnol. Prog. 10:472-479 (1994), disclose the cytoplasmic expression of r-K2S. The authors used an in vivo renaturation processes for the expressed r-K2S, which was purified from the cytoplasmic space of E. coli as inclusion body. Boehringer Mannheim use a similar cumbersome denaturing/refolding process involving the steps of cell digestion, solubilization under denaturing and reducing conditions and reactivation under oxidizing conditions in the presence of GSH/GSSG which is not cost effective and requires mutation of the amino acid sequence (Martin, U., et al., Z. Kardiol. 79:167-170 (1990)).
In 1991, Waldenström, M., et al., Gene 99:243-248 (1991), constructed a vector (pEZZK2P) for the secretion of kringle 2 plus serine protease domain to E. coli culture supernatant. Hydroxylamine was used to remove the ZZ fusion peptide from IgG-Sepharose purified fraction. The cleavage agent hydroxylamine required modification of the cleavage sites of kringle 2 plus serine protease (Asn177→Ser and Asn184→Gln) thus to protect it from hydroxylamine digestion. However, the resulting non-native, not properly folded K2S molecule is not suitable for therapeutic purposes. The unusual sequence may even activate the human immune system.